The long-term goal of these studies if to determine how the survival of spermatogenic cells is regulated by the Bcl-2 family of onoproteins. Although the potential exists for multiple influences on the regulation of mitotic events in the proliferating germ cells, a significant body of data has established a correlation between the size of the spermatogenic compartment and the number of early germ ells that do not survive to the haploid stage. We recently identified bcl-x as a member of the bcl-2 family of oncogenes preferentially expressed in the rat testis. Studies in this proposal concentrate on the regulation of the expression of two protein isoforms, Bcl-xL and Bcl-xS, and their roles in determining survival versus death of germ cells. Our studies will focus on the unique features of bcl-x expression in the seminiferous tubule. First, the bcl-x gene will be cloned and potential regulatory elements identified in its proximal promoter by DNA sequence analysis. Second, the specific regulation of the isolforms of bcl-x mRNA by androgens and stem cell growth factor will be evaluated using primary culture models of highly purified spermatogonia and early spermatocytes. Third, the stage specificity of bcl-x expression in the seminiferous tubule will be determined and the effects of stem cell factor and androgens on bcl-x expression and DNA synthesis in premeiotic germ cells ascertained. Fourth, the effects of changes in the ratio of the members of the Bcl-2 family of oncoproteins on programmed cell death in spermatogonia will be examined. Specific alterations in the balance of suppression versus induction of cell death will be accomplished by specific inhibition of Bcl-xL mRNA using an antisense oligonucleotide approach and by overexpression of Bcl-2 in transfected germ cells. Characterization of germ cell development and apoptotic processes in the bcl-2 transgenic mouse will facilitate studies to delineate the role of the bcl-2 family of genes in the survival of the male germ cells. Since testicular biopsies rom infertile males often show decreased numbers of proliferating and developing germ cells, we anticipate that this research will lead to significant new data on the relevance of apoptosis and its regulation to human male fertility.